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OPEN
Received: 18 October 2017
Accepted: 1 May 2018
Published: xx xx xxxx
ScIEntIfIc Reports | (2018) 8:7953 | DOI:10.1038/s41598-018-26308-5
Effect of Plant Density, Boron
Nutrition and Growth Regulation
on Seed Mass, Emergence and
Offspring Growth Plasticity in
Cotton
Ali Zohaib   1, Tahira Tabassum1, Abdul Jabbar1, Shakeel Ahmad Anjum1, Tasawer Abbas2,
Azhar Mehmood3, Sohail Irshad4, Muhammad Kashif5, Mohsin Nawaz1, Naila Farooq6,
Irfan Rasool Nasir7, Tassadduq Rasool1, Mubashar Nadeem1 & Riaz Ahmad1
Seed nutrients reserves have direct relationship with seed functional traits and influence offspring
performance. Effects of plant density, foliage boron (B) nutrition and mepiquat chloride (MC) growth
regulation on seed nutrients reserves, seed mass and production, and emergence and offspring growth
traits of cotton were studied in two years field experiment. Seed nutrients reserves and seed mass were
decreased at higher maternal plant density relative to lower plant density with concomitant decrease
in emergence and offspring seedling growth. However, maternal foliage B nutrition and MC growth
regulation enhanced seed nutrients reserves, seed mass, emergence and offspring seedling growth
performance. There was a significant positive relationship between seed mass and seed nutrients
reserves indicating that changes in nutrient availability/uptake in response to maternal ecological
factors determine variation in seed functional traits. Nonetheless, seed mass was positively correlated
with emergence percentage and negatively with emergence timing. Furthermore, variation in offspring
seedling growth traits with seed mass indicated the significance of initial seed nutrients reserves for
early seedling vigour and establishment. In conclusion, lower maternal plant density, B nutrition and
MC growth regulation ensued in higher emergence and offspring seedling growth of cotton because of
higher seed nutrient reserves and seed mass.
Effects of seed size (mass) on germination and seedling establishment of perennial plants in grassland1 and forest2
ecosystems have been well understood. Seed size is correlated with various seed functional traits, and often affects
the timing of germination, germination capacity, offspring seedling establishment, and diversity and composi-
tion of community1,3,4. Although seed mass has a great importance in natural ecosystems, its significance cannot
be overwhelmed in agro-ecosystems where seed size is the primary determinant of ecosystem productivity and
successful establishment of succeeding generation5. However, studies on the effects of ecological factors (plant
competition, nutrition, precipitation, temperature, light etc.) on seed mass, subsequent germination and seedling
establishment of cultivated annual crops in agro-ecosystems are sparse and not well understood6,7.
Cotton (Gossypium hirsutum L.) is cultivated as an annual crop, its perennial nature exists predominantly
under environmental factors (high precipitation, temperature and nutrient inputs etc.) that favour the vegetative
growth8. This often consequences in greater amounts of photosynthates and nutrients to be partitioned to vegeta-
tive parts while the decreased partitioning and remobilization of reserves to developing fruits become the source
1
Department of Agronomy, University of Agriculture, Faisalabad, 38040, Pakistan. 2Department of Agronomy,
College of Agriculture, University of Sargodha, Sargodha, 40100, Pakistan. 3Agronomy Department, University
College of Agriculture and Environmental Sciences, The Islamia University of Bahawalpur, Bahawalpur, 63100,
Pakistan. 4Agricultural Training Institute, Rahim Yar Khan, 64200, Pakistan. 5Department of Botany, University of
Agriculture, Faisalabad, 38040, Pakistan. 6Institute of Soil and Environmental Sciences, University of Agriculture,
Faisalabad, 38040, Pakistan. 7Adaptive Research Farm, Dera Ghazi Khan, 32200, Pakistan. Correspondence and
requests for materials should be addressed to A.Z. (email: alizohaib208@gmail.com)
1
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of variation in seed development and size9. Studies on natural ecosystems and perennial plants have revealed that
a significant correlation exists between maternal environment and seed development and seed mass10–12.
Plant competitiveness, soil and plant nutrient status, and alteration in the ability of plants to acquire nutrients
and their translocation to seeds can alter the pattern of seed development, seed mass, productivity and subse-
quent germination and seedling establishment in succeeding generation1,2,13–15. The plant nutrient status varies
with plant species and varieties, and is determined by total nutrient supply in soil and on the factors controlling
the nutrient availability to plants. Positive and regulatory effects of the seed mass are more often impossible to
distinguish from those of the seed mineral nutrients reserves16. A greater concentration of macronutrients in
surrounding ecological niche has often ensued in the production of heavier seeds having greater concentrations
and reserves of N and P17. A recent study on perennial plant Peucedanum oreoselinum from Asteraceae family
corroborated that a significant correlation existed between maternal habitat soil and seed macronutrients (N, P
and K) contents, and seed mass and seed macronutrients contents indicating that the positive effects of seed size
are more often dependent on nutrient availability and seed nutrient reserves2.
Plant density induces changes in the pattern of plant establishment, performance and productivity of an eco-
system, and success in the establishment of next generation because of complex relationship of inter-plant com-
petition within a population and community18. An agro-ecosystem is characterized by inclusion of intensive
external inputs. Thus manipulation in planting density can alter management decisions to acquire potential ben-
efits from ecosystem services. Increasing the plant density enhances the inter-plant competition owing to limited
plant available resources. Plant density modifies the plant canopy structure, plant micro environment (relative
humidity, canopy temperature and light transmittance)19 and capability of plants to acquire mineral nutrients
from the immediate rhizosphere owing to modulation in root growth due to competition for space20. Moreover,
the seed mass is influenced due to variation in plant density. Zhi et al.5 observed higher seed number per boll with
concomitant increase in seed size and vigour index at lower plant density of cotton while there was a progressive
decrease in seed area (density) and vigour index with increasing plant density.
Boron is an essential micronutrient that significantly affects the seed development and quality7,21. A short
term B deficiency during microsporogenesis hinders the development of anthers, and adversely affects the pollen
viability and growth of pollen tube consequently leading to male sterility and poor seed set21–23. Boron nutrition
affects the seed nutrients contents through modulation in uptake and translocation of other nutrients as well24.
For example, B has positive interactions with N, P, K, Cu, Zn and Fe, whereas, negative interactions with Ca,
Mg25–27, and Mn28. Moreover, it is involved in photo-assimilation and assimilate partitioning, and directly or
indirectly affects the seed development and seed size7,29. Dordas7 and Dordas et al.30 observed that seed mass,
yield and germination quality of cotton and sugar beets was superior in B treated plants and it was correlated with
enhanced B concentration in reproductive tissues.
Exogenous application of plant growth regulators (PGRs) is a well-known strategy to improve the crop physi-
ological performance, nutrient dynamics within plant body and crop yield under normal and stressed conditions
in agro-ecosystem8,31–33. Some studies have provided the ground basis that exogenously applied PGRs improve
the nutrient uptake and accumulation within the plant body34,35. Mepiquat chloride (1,1-dimethylpiperidinium
chloride) is a gibberellic acid (GA) inhibitor36 and is used worldwide to suppress excessive vegetative growth of
cotton by decreasing the plant height, internodes length, leaf area and length of fruiting and vegetative branches,
and to hasten crop maturity and avoid the yield losses37–45. Apart from manipulation of plant canopy structure,
MC enhances the cotton root growth by increasing number of lateral roots and increases the root vigor through
increased reducibility and respiratory rate thereby improving the nutrient uptake and partitioning within the
plant body14,38,46,47. Sawan et al.6 and Yang et al.14 noticed an increase in uptake and translocation of N and K to
cotton seed in MC treated plants.
Effects of plant density, foliage B nutrition and MC growth regulation on seed nutrients reserves, seed traits
and offspring growth performance were studied in two years multi-factor field experiment in agro-ecosystem.
The objectives of the study were to (1) quantify the single and interaction effects of plant density, B nutrition and
plant growth regulation on degree of variation in cotton seed nutrients reserves, seed mass and production, emer-
gence and offspring plasticity in seedling growth (2) to elucidate relative changes in seed nutrients reserves and
seed mass in response to maternal ecological factors and draw possible relationship (3) determine the quantitative
relationship between seed mass and cotton seed production (4) examine the seedling emergence, emergence time
and growth plasticity of cotton in next generation in relation to seed mass.

Results
Seed mass and seed production.  The lower plant density produced greater individual seed mass of cot-
ton as compared to higher plant density, being 3.5 and 4.6% higher during 2014 and 2015, respectively (Table 1).
Foliage B nutrition enhanced the seed mass as compared to control; however, there was no significant difference
between 600 mg/L and 1200 mg/L B. The heaviest seeds were produced by 1200 mg/L B nutrition, as compared
to control (6.2 and 6.4% higher during 2014 and 2015, respectively) (Table 1). Likewise, single applications of
MC at squaring and flowering increased the seed mass as compared to control but without significant difference
between both stages of treatment. Mepiquat chloride application at squaring stage caused highest increase in seed
mass (5.2 and 6% during 2014 and 2015, respectively). However, no significant interaction occurred regarding
seed mass (Table 1).
Cotton seed production ha−1 was higher by 16.3% during 2014 and 9.9% during 2015 at higher plant density
as compared to lower plant density (Table 1). Boron nutrition across both plant densities and MC treatments
improved the cotton seed production, as compared to control (13.5 and 12.8% during 2014 and 2015, respec-
tively) (Table 1). Mepiquat chloride across all plant density and B treatments significantly improved the seed
production, as compared to control; with greatest increase caused by MC treatment at squaring stage (15% during
2014 and 13.6% during 2015) (Table 1). During both years significant interactions of B with MC and B with plant

ScIEntIfIc Reports | (2018) 8:7953 | DOI:10.1038/s41598-018-26308-5 2

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Individual cotton seed mass (mg) Cotton seed production (kg ha−1)
Treatments 2014 2015 2014 2015
Density (D) (plants ha−1)
53333 76.3 ± 1.2 A 76.1 ± 1.1 A 1580 ± 28 B 1515 ± 25 B
88888 73.6 ± 0.7 B 72.6 ± 0.8 B 1838 ± 33 A 1665 ± 27 A
Boron nutrition (B)
Control 72.5 ± 1.1 B 71.8 ± 1.1 B 1599 ± 37 C 1489 ± 28 C
600 mg/L B 75.3 ± 0.9 A 74.9 ± 1.0 A 1713 ± 34 B 1601 ± 26 B
1200 mg/L B 77.0 ± 1.3 A 76.4 ± 1.9 A 1815 ± 57 A 1680 ± 40 A
Mepiquat chloride (MC)
Control 72.8 ± 0.9 B 71.9 ± 1.0 B 1583 ± 35 C 1481 ± 22 C
Squaring 76.6 ± 1.3 A 76.2 ± 1.9 A 1821 ± 49 A 1682 ± 36 A
Flowering 75.4 ± 0.7 A 75.1 ± 1.1 A 1723 ± 44 B 1607 ± 33 B
Source of variation (Pr > F)
D 0.004 0.002 <0.001 <0.001
B 0.001 0.002 <0.001 <0.001
MC 0.004 0.004 <0.001 <0.001
D × B 0.808 0.917 0.042 0.044
D × MC 0.912 0.947 0.799 0.749
B × MC 0.809 0.956 0.010 0.033
D × B × MC 0.941 0.903 0.874 0.977

Table 1.  Individual cotton seed mass and cotton seed production as influenced by plant density, foliar boron
nutrition and mepiquat chloride application. Values are mean ± SE of three replications. Means in a column
followed by different letters are significantly different at P < 0.05. Interactions with P < 0.05 are italicized to
indicate their significance.

density were observed, pertaining to cotton seed production (Table 1). Cotton seed production was greatest when
1200 mg/L B was applied in combination with MC application at squaring stage (Fig. 1a,b). Likewise, B nutrition
at 1200 mg/L at higher plant density led to the production of highest cotton seed (Fig. 1c,d).
A significant linear relationship between individual seed mass and seed production per plant occurred as
affected by plant density, B nutrition and MC plant growth regulation, during both years. The linear relationship
between seed mass and seed production per plant indicates that seed production varied uniformly by mean indi-
vidual seed mass (Fig. 2).

Seed nutrients reserves.  Seed primary macronutrients (N, P and K) and micronutrients (B, Zn, Fe and
Mn) reserves significantly differed between plant densities. Higher seed N (2.1 and 2.2%), P (5.1 and 8.2%), K (9.9
and 8.4%), B (19.9 and 20.5%), Zn (5.4 and 6.7%), Fe (4 and 3.6%) and Mn (8.6 and 7%) contents were produced
at lower plant density as compared to higher plant density during 2014 and 2015, respectively (Tables 2 and 3).
Significantly greater seed N (4.7 and 4.5%), P (8.3 and 12.1%), K (12.1 and 10.4%), B (31.6 and 36.4%), Zn (11.4
and 10%) and Fe (14.3 and 11.2%) reserves were observed in response to foliage B nutrition across MC and plant
density treatments as compared to control, during 2014 and 2015, respectively. The P contents did not differ
between 600 and 1200 mg/L B during 2015. However, Mn contents were significantly decreased (10.4 and 11%)
with concomitant increase in B concentration during 2014 and 2015, respectively (Tables 2 and 3). Compared
with control, MC led to increase in seed nutrients reserves; although no significant differences occurred between
both treatment stages except K during 2015 and B during both years. Across B and plant density treatments the
greatest enrichment of seed with N (3.8 and 4.2%), P (8.3 and 12.1%), K (12.2 and 11%), B (18.2 and 17.8%), Zn
(9.2 and 8.2%) and Fe (3.4 and 4.9%) was caused by treatment of MC at squaring stage, during 2014 and 2015,
respectively. Seed Mn contents were decreased (5.8 and 4.7%) by MC application while there was no difference
between both stages of MC application (Tables 2 and 3).
Interactions of plant density, B and MC were non-significant for seed nutrients reserves (N, P, K, Zn, Fe and
Mn); except B for which the interaction effects of B with MC and B with plant density were significant, during
both years while interaction of MC with plant density was significant during 2015 (Tables 2 and 3). Seed B con-
tents were substantially increased by application of 1200 mg/L B plus MC application at squaring stage (Fig. 3a,b).
The B application at 1200 mg/L at lower plant density resulted in significantly higher seed B reserves (Fig. 3c,d).
Similarly, MC application at squaring stage on lower plant density caused the greatest improvement in seed B
contents during 2015 (Fig. 3f).
Seed nutrients contents (N, P, K, B, Zn and Fe) and individual seed mass were positively correlated in response
to plant density, foliar B nutrition and MC growth regulation, during both years (Table 4). This positive relation-
ship manifested that changes in seed mass and macro- and micronutrients contents were relatively uniform across
treatments (Table 4).

Emergence and seedling vigour.  Final emergence and seedling vigour index were significantly greater
from seed produced at lower plant density with concomitant decrease in mean emergence time. Higher final

ScIEntIfIc Reports | (2018) 8:7953 | DOI:10.1038/s41598-018-26308-5 3

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Figure 1.  Cotton seed production as affected by interaction effects of (a,b) boron with mepiquat chloride and
(c,d) boron with plant density. Bars are mean ± SE of three replications. Bars sharing same letter don’t differ
significantly at P < 0.05.

Figure 2.  Relationship between individual seed mass (mg) and seed production (g plant−1) during 2014
and 2015. Coefficient of determination (R2), significance of coefficient (P < 0.05) and dependence of seed
production (y) on individual seed mass (x) are given (n = 18).

emergence (6.3 and 5.9% during 2014 and 2015, respectively) occurred at lower plant density than higher plant
density (Table 5). Foliage B nutrition of maternal cotton plants significantly improved the emergence and seed-
ling vigour index. Final emergence was higher by 7.2 and 8.9 percentages during 2014 and 2015, respectively, by
1200 mg/L B nutrition, as compared to control. Moreover, compared with control, same B nutrition rate encoun-
tered a decrease in mean emergence time by 6.5 and 4.3% during 2014 and 2015, respectively (Table 5). Likewise,
MC application at squaring stage on maternal plants was superior in improving the final emergence (6.7 and 7.8%
during 2014 and 2015, respectively) and seedling vigour index (20.9 and 22.7% during 2014 and 2015, respec-
tively) (Table 5).
The interactions of plant density, B nutrition and MC application were non-significant for final emergence
and mean emergence time. However, the interaction effect of B with MC was significant for seedling vigour index

ScIEntIfIc Reports | (2018) 8:7953 | DOI:10.1038/s41598-018-26308-5 4

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Seed nitrogen (mg g−1 DW) Seed phosphorus (mg g−1 DW) Seed potassium (mg g−1 DW)
Treatments 2014 2015 2014 2015 2014 2015
Density (D) (plants ha−1)
53333 32.9 ± 0.23 A 32.1 ± 0.26 A 3.9 ± 0.04 A 3.71 ± 0.06 A 20.3 ± 0.37 A 19.0 ± 0.05 A
88888 32.2 ± 0.24 B 31.4 ± 0.22 B 3.7 ± 0.04 B 3.40 ± 0.05 B 18.3 ± 0.34 B 17.4 ± 0.02 B
Boron nutrition (B)
Control 31.8 ± 0.27 B 31.0 ± 0.32 B 3.6 ± 0.05 C 3.3 ± 0.06 B 18.2 ± 0.48 C 17.3 ± 0.31 C
600 mg/L 32.5 ± 0.26 AB 31.9 ± 0.27 AB 3.8 ± 0.04 B 3.6 ± 0.07 A 19.2 ± 0.40 B 18.3 ± 0.32 B
1200 mg/L 33.3 ± 0.29 A 32.4 ± 0.25 A 3.9 ± 0.05 A 3.7 ± 0.08 A 20.4 ± 0.49 A 19.1 ± 0.33 A
Mepiquat chloride (MC)
Control 31.9 ± 0.28 B 31.1 ± 0.25 B 3.6 ± 0.05 B 3.3 ± 0.06 B 18.1 ± 0.41 B 17.2 ± 0.29 C
Squaring 33.1 ± 0.27 A 32.4 ± 0.27 A 3.9 ± 0.05 A 3.7 ± 0.08 A 20.3 ± 0.47 A 19.1 ± 0.33 A
Flowering 32.6 ± 0.29 AB 31.8 ± 0.32 AB 3.8 ± 0.06 A 3.6 ± 0.07 A 19.5 ± 0.48 A 18.3 ± 0.32 B
Source of variation (Pr > F)
D 0.025 0.034 <0.001 <0.001 <0.001 <0.001
B 0.002 0.004 <0.001 <0.001 <0.001 <0.001
MC 0.008 0.006 <0.001 <0.001 <0.001 <0.001
D × B 0.913 0.875 0.480 0.865 0.455 0.841
D × MC 0.996 0.984 0.754 0.780 0.887 0.947
B × MC 0.957 0.998 0.715 0.167 0.821 0.941
D × B × MC 0.834 0.986 0.316 0.388 0.461 0.974

Table 2.  Cotton seed primary macronutrients reserves as influenced by plant density, foliar boron nutrition
and mepiquat chloride application. Values are mean ± SE of three replications. Means in a column followed by
different letters are significantly different at P < 0.05. Interactions with P < 0.05 are italicized to indicate their
significance.

during both years (Table 5). Substantially greater seedling vigour index was perceived by 1200 mg/L B plus MC
application at squaring stage, during both years (Fig. 4a,b). Linear regression manifested a linear increase in final
emergence with seed mass (Fig. 5a). However, there was a negative linear relation between seed mass and mean
emergence time indicating a linear decrease in mean emergence time with increase in seed mass (Fig. 5b).

Offspring seedling growth.  Seedling growth traits (root length, shoot length, root dry biomass and shoot
dry biomass) of cotton in succeeding generation were significantly greater at lower plant density as compared
to higher plant density (Table 6). Boron treatment on cotton plants effectively improved the offspring seedling
growth traits of cotton and the effect was greater at 1200 mg/L B than lower concentration (600 mg/L) and con-
trol (Table 6). Growth regulation by MC manifested a similar significant improvement in seedling growth traits
as compared to control, during both years (Table 6). The interactions for root length and shoot length were
non-significant; however, interactive effect of B with MC on root and shoot dry biomass was significant, during
both years (Table 6). The greatest root and shoot dry biomass accumulation occurred by B nutrition at 1200 mg/L
concentration in combination with MC treatment at squaring stage (Fig. 6c,d). There was a significant posi-
tive linear relationship between seedling growth traits and seed mass indicating that seedling growth traits were
increased uniformly with increase in seed mass (Fig. 7a–d).

Discussion
The results of this study have revealed new information that maternal plant density, foliage B nutrition and MC
growth regulation influence the offspring performance in terms of emergence and seedling growth plasticity in
annual cultivated cotton.

Seed nutrients reserves, seed mass and production.  Individual seed mass of cotton was decreased
at higher plant density while increased by foliage B nutrition and MC growth regulation (Table 1). This might
be attributed to variation in nutrient availability and uptake in response to maternal environmental variables as
indicated by differential seed nutrients reserves (Tables 2 and 3). Cotton seed nutrients reserves were increased at
higher plant density relative to lower plant density. However, in comparison with control, B and MC enhanced the
seed nutrients contents at both plant densities except Mn (Tables 2 and 3). This variation in cotton seed nutrients
reserves might be explained on the basis of root growth which is necessary for nutrient uptake48. The root growth
is depressed at higher plant density due to competition20 while increased by B49 and MC47 influencing the nutrient
dynamics within plants. Moreover, B and MC alters the ability of plants for nutrient uptake and translocation
through improved nutrient metabolism and assimilation24,50–52. However, decreased seed Mn content by B may
be attributed to their antagonistic effect with each other that led to decreased uptake and translocation53. Previous
studies have reported similar results that plant and seed nutrients contents decreased at high plant density15,20,
while increased in response to B nutrition24,26,27 and MC application6,14.
Although little attention has been given to the relationship between seed nutrients contents and seed size.
In this study, positive linear relationship between seed nutrients contents (N, P, K, B, Zn and Fe) and seed mass
(Table 4) manifested that large sized seeds were better provisioned due to greater availability of nutrients to

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Seed boron (µg g−1 DW) Seed zinc (µg g−1 DW) Seed iron (µg g−1 DW) Seed manganese (µg g−1 DW)
Treatments 2014 2015 2014 2015 2014 2015 2014 2015
Density (D) (plants ha−1)
53333 48.2 ± 1.1 A 41.5 ± 0.2 A 44.8 ± 0.56 A 44.7 ± 0.56 A 186.2 ± 2.35 A 170.7 ± 2.05 A 21.0 ± 0.30 A 21.4 ± 0.26 A
88888 38.6 ± 1.2 B 33.0 ± 0.2 B 42.4 ± 0.63 B 41.7 ± 0.53 B 178.8 ± 2.28 B 164.6 ± 2.12 B 19.2 ± 0.28 B 19.9 ± 0.31 B
Boron nutrition (B)
Control 36.7 ± 1.30 C 30.8 ± 0.99 C 41.3 ± 0.60 C 41.1 ± 0.67 C 169.6 ± 1.84 C 158.6 ± 2.34 C 21.2 ± 0.38 A 21.8 ± 0.30 C
600 mg/L 45.2 ± 1.52 B 38.8 ± 1.37 B 43.4 ± 0.55 B 43.4 ± 0.69 B 184.1 ± 1.74 B 168.1 ± 1.58 B 20.1 ± 0.31 B 20.7 ± 0.31 B
1200 mg/L 48.3 ± 1.44 A 42.0 ± 1.38 A 46.0 ± 0.75 A 45.2 ± 0.58 A 193.8 ± 1.81 A 176.3 ± 2.07 A 19.0 ± 0.38 C 19.4 ± 0.33 A
Mepiquat chloride (MC)
Control 39.5 ± 1.55 C 33.8 ± 1.26 C 41.4 ± 0.58 B 41.4 ± 0.67 B 178.9 ± 2.68 B 162.6 ± 2.43 B 20.7 ± 0.37 A 21.2 ± 0.39 A
Squaring 46.7 ± 1.86 A 39.8 ± 1.75 A 45.2 ± 0.71 A 44.8 ± 0.70 A 185.0 ± 2.91 A 170.6 ± 3.01 A 19.5 ± 0.40 B 20.2 ± 0.35 B
Flowering 44.1 ± 1.69 B 38.1 ± 1.70 B 44.2 ± 0.76 A 43.4 ± 0.69 A 183.6 ± 3.19 AB 169.8 ± 2.07 A 20.1 ± 0.43 AB 20.5 ± 0.40 AB
Source of variation (Pr > F)
D <0.001 <0.001 <0.001 <0.001 <0.001 0.002 <0.001 <0.001
B <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.001 <0.001
MC <0.001 <0.001 <0.001 <0.001 0.001 0.001 0.032 0.032
D × B 0.024 0.036 0.738 0.526 0.645 0.519 0.828 0.838
D × MC 0.128 0.034 0.999 0.967 0.764 0.653 0.746 0.744
B × MC 0.036 0.002 0.433 0.754 0.973 0.238 0.985 0.995
D × B × MC 0.649 0.134 0.530 0.835 0.874 0.940 0.952 0.841

Table 3.  Cotton seed micronutrients reserves as influenced by plant density, foliar boron nutrition and
mepiquat chloride application. Values are mean ± SE of three replications. Means in a column followed by
different letters are significantly different at P < 0.05. Interactions with P < 0.05 are italicized to indicate their
significance.

developing seeds. Previous studies have reported that seed provisioning is associated with nutrient availability
which may be influenced due to soil nutrient gradient2, competition among plants for available resources4 and/or
nutrient enrichment1. Plants often growing in nutrient rich environments produce large sized seeds containing
greater nutrient contents17,54. In this study, the strength of relationship between concentration of nutrients and
seed mass (Table 4) was strong enough to indicate that bigger seeds contained greater nutrients contents (mg per
seed) than the smaller seeds because of higher nutrient concentration in response to maternal ecological factors.
It is also evident from the calculations from seed mass and nutrients concentrations (Tables 1–3) that the seeds
produced at lower plant density with B nutrition and MC growth regulation had greater nutrients reserves than
seeds produced by plants at higher plant density and without B nutrition and MC growth regulation. Similarly,
Balestri et al.12 found higher C, N and P contents in bigger seeds of Posidonia oceanica than smaller ones.
In this study, individual seed mass was linearly related to seed production per plant of cotton (Fig. 2).
Therefore, although seed production per hectare was increased (Table 1) but seed production per plant was
decreased with increase in plant density due to decreased individual seed mass. However, seed production per
plant and per hectare was increased by exogenous application of B and MC which was due to linear increase in
seed mass (Fig. 2; Table 1). Similarly, N and P enrichment of maternal plants of Stipa krylovii and Artemisia frigida
produced heavier seeds in temperate steppe ecosystem. Furthermore, a significant positive relationship existed
between seed mass and seed production1.

Emergence and emergence timing.  There was a positive linear relationship of seed mass with final
emergence and emergence timing of cotton in response to plant density, B nutrition and MC growth regulation
(Fig. 5a,b). The results of this study are consistent with the fact that heavier seeds germinate rapidly and gives
better seed germination55. This indicates that bigger seeds having greater seed nutrients reserves are advantageous
pertaining to emergence and emergence timing, and have more time for the seedling establishment. Moreover,
lower seedling vigour index of seed from plants at higher plant density and higher seedling vigour index of seed
from B and MC treated plants (Fig. 4a,b; Table 5) indicates that higher amounts of seed nutrients reserves in large
sized seeds enhanced the emergence and emergence timing, and had better seedling establishment than smaller
seeds with less nutrients reserves. Our results are consistent with previous reports that seed mass has relation-
ship with germination and seedling vigour. van Mölken et al.3 observed that higher germination percentage was
produced by heavier seeds of Tragopogon pratensis subsp. pratensis from Asteraceae family. Ninh et al.56 reported
positive relationship between seed size and germination percentage of Angelica acutiloba. Yanlong et al.55 reported
that large sized seeds of Ligularia virgaurea had higher germination, germination rate and seedling vigour under
both light and shade conditions.

Offspring growth plasticity.  The seedling growth of offspring significantly differed by the influence of
plant density, B nutrition and MC growth regulation. The seeds from plants at higher plant density produced
seedlings with lesser root and shoot length, and root and shoot dry biomass. On the other hand, seedlings from
seeds produced by B and MC treated plants had greater root and shoot length as well as dry biomass, as compared

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Figure 3.  Cotton seed boron content as affected by interaction effects of (a,b) boron with mepiquat chloride,
(c,d) boron with plant density and (e,f) mepiquat chloride with plant density. Bars are mean ± SE of three
replications. Bars sharing same letter don’t differ significantly at P < 0.05.

to untreated control (Fig. 6a–d; Table 6). This growth plasticity can be explained by the fact that large sized seeds
with greater nutrients reserves produce vigorous seedlings due to early head start and greater energy available2.
It was also evident from significant linear relationship between seedling growth traits and seed mass (Fig. 7a–d)
that bigger seeds produced more successful seedlings with more competitive ability than smaller seeds. The vig-
orous seedlings produced from bigger seeds with greater nutrients reserves have more competitive ability over
seedlings from small seeds with less reserves57 which is advantageous especially in agro-ecosystems where higher
competitive ability of crop plants is beneficial in improving the system productivity with minimum inclusion of
external inputs.
Results of this study suggest that offspring growth performance is affected more by seed nutrients reserves
rather than seed phenotypic traits. This is because firstly, the seed phenotypic traits especially seed size is affected
by nutrient availability2,4,17,57, as indicated by positive relationship between seed nutrients reserves and individ-
ual seed mass in this study. Secondly, increase in seed size (mass) may be the result of increased mass of seed
structures (seed coat, awns etc.) rather than seed embryo or endosperm1,58. Moreover, in this study a significant
negative relationship between seed mass and mean emergence time (Fig. 5b) indicated that the seedlings from
larger seeds started early vigorous growth and still had more time to develop due to greater amounts of nutri-
ents reserves. Yanlong et al.55 reported that seedlings of L. virgaurea produced from larger seeds had greater dry

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Individual seed mass (mg)

Variables 2014 2015
Seed nitrogen content (mg g−1 DW) 0.9146** 0.9634**
**
Seed phosphorus content (mg g −1
DW) 0.9456 0.9357**
**
Seed potassium content (mg g −1
DW) 0.9372 0.9652**
**
Seed boron content (mg g −1
DW) 0.9428 0.9449**
Seed zinc content (mg g−1 DW) 0.9270** 0.9242**
Seed iron content (mg g−1 DW) 0.8686** 0.8713**
Seed manganese content (mg g−1 DW) −0.3183NS −0.3345NS

Table 4.  Pearson’s coefficients of correlation between individual seed mass and seed nutrients (N, P, K, B, Zn,
Fe and Mn) contents as influenced by plant density, foliar boron nutrition and mepiquat chloride application
(n = 18). **P < 0·01; *P < 0·05; NSnon-significant.

Final emergence (%) Mean emergence time (days) Seedling vigour index
Treatments 2014 2015 2014 2015 2014 2015
Density (D) (plants ha−1)
53333 66 ± 2 A 71 ± 2 A 4.6 ± 0.1 A 4.9 ± 0.0 B 1860 ± 49 A 1808 ± 48 A
88888 60 ± 1 B 65 ± 2 B 4.6 ± 0.2 A 5.0 ± 0.1 A 1576 ± 39 B 1562 ± 39 B
Boron nutrition (B)
Control 59 ± 1 B 63 ± 2 B 4.8 ± 0.0 A 5.1 ± 0.1 A 1526 ± 40 C 1484 ± 37 C
600 mg/L 63 ± 3 AB 69 ± 2 A 4.5 ± 0.1 B 4.9 ± 0.1 AB 1735 ± 53 B 1726 ± 55 B
1200 mg/L 67 ± 2 A 72 ± 4 A 4.5 ± 0.0 B 4.9 ± 0.0 B 1893 ± 64 A 1845 ± 56 A
Mepiquat chloride (MC)
Control 59 ± 2 B 64 ± 2 B 4.7 ± 0.2 A 5.0 ± 0.1 A 1537 ± 40 C 1509 ± 41 C
Squaring 66 ± 4 A 72 ± 4 A 4.5 ± 0.1 B 4.9 ± 0.0 A 1858 ± 62 A 1852 ± 57 A
Flowering 64 ± 2 AB 68 ± 3 AB 4.5 ± 0.1 B 4.9 ± 0.1 A 1759 ± 63 B 1694 ± 56 B
Source of variation (Pr > F)
D <0.001 0.004 0.487 0.014 <0.001 <0.001
B 0.001 0.002 <0.001 0.035 <0.001 <0.001
MC 0.003 0.008 <0.001 0.173 <0.001 <0.001
D × B 0.940 0.963 0.166 0.848 0.545 0.114
D × MC 0.780 0.768 0.856 0.804 0.561 0.497
B × MC 0.908 0.921 0.600 0.783 0.009 0.039
D × B × MC 0.908 0.961 0.976 0.991 0.229 0.093

Table 5.  Emergence, emergence time and seedling vigour index of cotton from seed produced by plants in
response to plant density, foliar boron nutrition and mepiquat chloride application. Values are mean ± SE
of three replications. Means in a column followed by different letters are significantly different at P < 0.05.
Interactions with P < 0.05 are italicized to indicate their significance.

biomass. Similarly Kolodziejek2 observed that maternal enrichment of S. krylovii and A. frigida with N and P
produced heavier seeds that led to vigorous seedlings with higher initial growth.
In summary, plant density, B nutrition and MC plant growth regulation significantly affected the seed mass of
cotton and seed nutrients reserves (N, P, K, B, Zn and Fe). The positive linear relationship between seed nutrients
reserves and seed mass indicated that nutrient availability and/or uptake influence the seed phenotypic traits in
response to maternal ecological factors. The variation in seed mass ultimately influenced the cotton seed produc-
tion, offspring emergence and seedling growth plasticity. The results suggest that lower maternal plant density,
B nutrition and MC growth regulation results in higher emergence and offspring seedling growth due to higher
seed mass and nutrients reserves. This improved emergence and competitive ability of offspring seedlings of cot-
ton through better management practices could be exploited to improve the agro-ecosystem productivity while
reducing the input cost (e.g. seed, fertilizer, herbicide etc.).

Methods
Field experimental site.  A 2-years field experiment was conducted at Agronomic Research Area, University
of Agriculture, Faisalabad (31.25°N, 73.06°E and 183 masl), Pakistan, during 2014 and 2015. Soil samples were
collected prior to sowing the crop using auger from a depth of 0–30 cm from different locations of field to deter-
mine the physico-chemical properties of soil, during both years. The soil was alkaline-calcareous, B deficient and
sandy loam in nature with high pH (8.2, 8.1), calcium carbonate (5.20, 5.11%) and electrical conductivity (1.78,
1.72 dS m−1), while lower in organic matter (1.12, 0.97%). The soil was medium in nutrient status with lower

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Figure 4.  Seedling vigour index of cotton seedlings from seed produced by plants in response to interaction
effect of foliar boron nutrition with mepiquat chloride application. Bars are mean ± SE of three replications.
Bars sharing same letter don’t differ significantly at P < 0.05.

Figure 5.  Relationship between (a) individual seed mass (mg) (mean) and final emergence (%), and (b)
seed mass (mg) and mean emergence time (days) during 2014 and 2015. Coefficients of determination (R2),
significance of coefficients (P < 0.05) and dependence of (a) final mergence percentage (y) and (b) mean
emergence time (y) on individual seed mass (x) are given (n = 18).

total N (0.050, 0.049%), available P (7.09, 6.91 ppm), exchangeable K (259, 248 ppm), B (0.47, 0.49 ppm), Zn
(1.70, 1.50 ppm) and Fe (5.21, 5.10 ppm) during both years, respectively. The soil of experimental site belongs to
Lyallpur soil series (aridisol fine silty, mixed, hyperthermic Ustalfic), Haplarged in the USDA classification and

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Root length (cm) Shoot length (cm) Root dry biomass (mg) Shoot dry biomass (mg)
Treatments 2014 2015 2014 2015 2014 2015 2014 2015
Density (D) (plants ha−1)
53333 7.3 ± 0.4 A 6.9 ± 0. A 20.72 ± 0.8 A 18.7 ± 0.5 A 17.9 ± 1.0 A 14.6 ± 0.4 A 206.7 ± 3.9 A 179.4 ± 3.4 A
88888 6.8 ± 0.1 B 6.4 ± 0.1 B 19.47 ± 0.3 B 17.8 ± 0.3 B 14.7 ± 0.3 B 13.1 ± 0.2 B 186.6 ± 3.1 B 165.0 ± 3.0 B
Boron nutrition (B)
Control 6.6 ± 0.2 B 6.2 ± 0.1 B 19.05 ± 0.3 B 17.5 ± 0.2 B 14.9 ± 0.6 C 12.8 ± 0.2 C 181.9 ± 2.9 C 159.2 ± 2.5 C
600 mg/L 7.1 ± 0.1 A 6.7 ± 0.2 A 20.27 ± 0.4 A 18.4 ± 0.8 AB 16.6 ± 0.7 B 13.9 ± 0.3 B 200.0 ± 4.5 B 173.5 ± 3.5 B
1200 mg/L 7.4 ± 0.4 A 6.9 ± 0.2 A 20.97 ± 0.9 A 18.8 ± 0.3 A 17.4 ± 1.1 A 14.7 ± 0.3 A 208.1 ± 5.0 A 183.9 ± 4.5 A
Mepiquat chloride (MC)
Control 6.7 ± 0.2 B 6.2 ± 0.1 B 19.20 ± 0.3 B 17.5 ± 0.3 B 15.2 ± 0.4 B 12.9 ± 0.2 C 181.6 ± 2.9 B 158.9 ± 2.2 B
Squaring 7.3 ± 0.4 A 6.9 ± 0.2 A 20.79 ± 0.5 A 18.9 ± 0.7 A 17.1 ± 0.7 A 14.7 ± 0.4 A 207.0 ± 4.9 A 180.9 ± 4.0 A
Flowering 7.2 ± 0.2 A 6.7 ± 0.1 A 20.30 ± 0.4 AB 18.3 ± 0.3 AB 16.6 ± 0.5 A 14.0 ± 0.3 B 201.4 ± 4.6 A 176.8 ± 4.4 A
Source of variation (Pr > F)
D <0.001 <0.001 0.002 0.022 <0.001 <0.001 <0.001 <0.001
B <0.001 <0.001 0.001 0.019 <0.001 <0.001 <0.001 <0.001
MC 0.001 <0.001 0.005 0.010 <0.001 <0.001 <0.001 <0.001
D × B 0.965 0.119 0.918 0.738 0.553 0.671 0.114 0.912
D × MC 0.635 0.169 0.966 0.745 0.820 0.934 0.417 0.793
B × MC 0.556 0.372 0.574 0.925 0.005 0.029 0.021 0.012
D × B × MC 0.964 0.192 0.966 0.902 0.786 0.989 0.515 0.235

Table 6.  Seedling traits (root length, shoot length, root dry biomass and shoot dry biomass) of cotton from
seed produced by plants in response to plant density, foliar boron nutrition and mepiquat chloride application.
Values are mean ± SE of three replications. Means in a column followed by different letters are significantly
different at P < 0.05. Interactions with P < 0.05 are italicized to indicate their significance.

Haplic Yermosols in the FAO classification. The meteorological data during both experimental seasons are given
in Table 7.

Field experimental design.  This study was accomplished in two phases. In first phase, 2-year field experi-
ment was conducted. The experiment was carried out using randomized complete block design with three-factor
factorial arrangement including three replications. Net plot size was 6 m × 3 m. The treatment variables were two
plant densities [lower plant density (53333 plants ha−1) and higher plant density (88888 plants ha−1)], foliage
applied B (0, 600 and 1200 mg/L B) and single foliar applications of MC solution with 70 mg/L concentration at
two different growth stages viz. squaring and flowering. Control plants received water spray. The B was foliage
applied five weeks after sowing using boric acid (17% B); while, MC [98% SP from Henan Haoyuhang Economic
and Trade Co., Ltd] was foliar applied using a Knapsack hand sprayer at a pressure of 207 kPa. The spray volume
for MC application at squaring was 300 L ha−1 while for B and MC application at flowering stage was 350 L ha−1.

Field management.  Cotton cultivar MNH-886 (Containing Bt gene, developed by Central Cotton Research
Institute, Multan, Pakistan) was sown on May 22nd during 2014 and May 25th during 2015. Prior to sowing, the
scarification treatment of seed was carried out with commercial sulfuric acid (1:10 ratio of seed and acid) and
treated with fungicide (dynasty CST 125 FS @ 3 g kg−1 seed). The sowing was carried out at beds using dibbler
and keeping the inter row spacing of 75 cm while intra row spacing was varied according to the treatments i.e. 25
and 15 cm to maintain plant densities of 53333 and 88888 plants ha−1, respectively. Seed rates of 15 and 25 kg ha−1
was used for attaining plant densities of 53333 and 88888 plants ha−1, respectively and 2–3 seeds were sown per
hill. Thinning was carried out 25 DAS to keep the desired plant densities. Fertilization was carried out at the rate
of 200-120-110 kg NPK ha−1 using urea (46% N), diammonium phosphate (18% N: 46% P2O5) and sulfate of
potash (50% K2O), during both years. All P, K and 1/3rd N was applied at sowing; while, remaining N was applied
at squaring and boll formation stages in equal splits. The crop was irrigated eight times during 2014 and five
times during 2015 besides irrigation applied at sowing keeping the crop and weather conditions in consideration.
During second year, occurrence of heavy rainfall decreased the irrigation requirements (Table 7). Plant protection
measures were carried out according to the local recommendations in order to keep the weeds, insect pests and
diseases below economic threshold level.

Measurements of seed mass and seed nutrients reserves.  The crop was harvested in two manual
pickings. The seed cotton obtained from all pickings was mixed and ginned using roller type, hand-fed laboratory
gin. Cotton seed production (kg ha−1) was determined after ginning. Individual cotton seed mass was determined
by taking the mean mass of 100 seeds collected randomly after ginning. The delinted seed samples were assayed
to determine the concentration of macro and micronutrients. The delinted seed samples were dried under sun
and electric oven at 70 °C for 24 h, ground to powder form (Cyclotec 1093 Sample Mill, Sweden) to pass through a
30-mesh screen. The samples were digested using sulfuric acid and catalyst mixture and the N was determined by
Kjeldhal distillation method as described by Estefan et al.59. The P was analyzed by ammonium-vanadomolybdate

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Figure 6.  Seedling traits [(a,b) root dry biomass and (c,d) shoot dry biomass] of cotton from seed produced
by plants in response to interaction effect of foliar boron nutrition and mepiquat chloride application. Bars are
mean ± SE of three replications. Bars sharing same letter don’t differ significantly at P < 0.05.

colorimetric method at 410 nm using the same extract59. For determination of K and micronutrients viz. Zn, Fe
and Mn the samples were soaked over-nightly in di-acid mixture and digested on block digester. The K was deter-
mined by flame photometer using the method of Chapman and Pratt60; while, Zn, Fe and Mn were assayed using
the atomic absorption spectrometer according to the method described by Estefan et al.59. Boron was determined
by dry ashing the ground seed material in muffle furnace at 550 °C for 6 h60. The ash was taken in 0.36N H2SO4
and the B concentration was determined by spectrophotometer at 420 nm wavelength using azomethine-H col-
orimetric method61. The contents of seed macronutrients (N, P and K) was expressed as mg g−1 dry weight (DW)
and micronutrients (B, Zn, Fe and Mn) as µg g−1 DW.

Soil bioassay.  In second phase, the seed collected from maternal plants was subjected to soil bioassay to
assess the emergence and offspring plasticity in seedling growth. The bioassay was not carried out immediately
after collection of seed, instead seeds were stored at room temperature (25 °C) to avoid effects of dormancy and
poor seed germination. The bioassay was performed in subsequent year every time after collection of seed i.e.
2015 and 2016 (however, hereafter we shall call the years 2015 and 2016 as 2014 and 2015, respectively, to avoid
confusion). The experiment was conducted by using completely randomized design with factorial arrangement
and three replications. Ten seeds were sown in soil filled pots (25 cm diameter and 20 cm height) following scar-
ification treatment with sulfuric acid (1:10 ratio of seed and acid). At the start 150 mL of deionized water was
applied to each pot and then according to the requirement. The experiments were observed for 30 days. The
average maximum temperature was 31.1 and 33.0 °C, and minimum temperature was 19.3 and 19.7 °C during first
and second year, respectively, during the period of experiment.

Measurements of emergence and seedling growth.  The seedlings emerged were counted daily up to
the end of experiment as per method of Association of Official Seed Analysts62. Final emergence percentage was
calculated by using following the formula;
No . of seedlings emerged
Final emergence(%) = × 100
Total number of seeds (1)
Equation of Ellis and Roberts63 was used to calculate the mean emergence time;

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Figure 7.  Relationship between (a) individual seed mass (mg) and root length (cm), (b) individual seed mass
(mg) and shoot length (cm), (c) individual seed mass (mg) and root dry biomass (mg), and (d) individual seed
mass (mg) and shoot dry biomass (mg) during 2014 and 2015. Coefficients of determination (R2), significance
of coefficients (P < 0.05) and dependence of (a) root length (y), (b) shoot length (y), (c) root dry biomass (y)
and (d) shoot dry biomass (y) on individual seed mass (x) are given (n = 18).

Temperature (°C)
Total rainfall Relative Monthly Monthly Sunshine
(mm) humidity (%) maximum minimum Daily mean (h)
Month 2014 2015 2014 2015 2014 2015 2014 2015 2014 2015 2014 2015
May 41.2 17.0 33.2 27.5 36.6 38.7 23.7 24.9 30.1 31.8 10.4 10.4
June 7.1 11.6 33.5 39.0 40.9 38.0 28.1 25.6 34.5 31.8 9.4 9.4
July 57.5 128.0 53.9 61.1 37.0 34.9 28.0 27.0 32.5 31.0 9.0 5.1
August 4.8 48.4 52.7 60.4 37.1 35.9 27.3 26.7 32.2 31.3 9.1 7.0
September 140.2 75.2 61.2 51.6 33.9 35.4 24.5 24.4 29.2 29.9 7.7 8.2
October 3.6 14.5 54.6 52.9 31.3 32.2 19.1 19.1 25.2 25.4 7.8 8.3
November 10.0 8.8 61.7 61.5 26.3 27.1 11.5 12.1 18.9 19.6 7.6 6.6

Table 7.  Meteorological conditions during cotton growing seasons of field experiment. Source: Agro-
meteorology Cell, Department of Crop Physiology, University of Agriculture, Faisalabad, Pakistan.

∑Dn
Mean emergence time (days) =
N (2)
where, n is the number of emerged seedlings on day D and N is the total number of germinated/emerged seeds.
At the end of experiment, seedlings were uprooted and their shoot and root lengths were measured. Shoots
and roots were separated and dried in oven at 70 °C till constant weight to measure the dry biomass accumulation.
Seedling vigour index (SVI) was calculated using the equation of Abdul-Baki and Anderson64:
SVI = Final emergence (%) × Seedling length (cm) (3)

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Statistical analyses.  Rainfall, air temperature and accumulation of heat units were different in 2014 and
2015 from May through November at field experimental site, therefore data of each year was analyzed separately.
All the data were analyzed using the Univariate General Linear Models (GLM) procedure of SPSS 19. The plant
density, B and MC were entered as fixed factors while replication (block) was entered as random factor. The treat-
ments’ means were compared by using the Tukey’s honest significance difference (HSD) test at 5% probability.
Pearson’s coefficients were calculated to determine the correlation between seed mass and seed nutrients contents
(N, P, K, B, Zn, Fe and Mn) to test the effect of plant density, B nutrition and MC growth regulation on seed
nutrients and seed mass. Linear regression analyses were performed to determine the quantitative relationship
of individual seed mass with seed production, final emergence, mean emergence time and seedling growth traits.

References
1. Li, Y., Hou, L., Song, B., Yang, L. & Li, L. Effectsof increased nitrogen and phosphorus deposition on offspring performance of two
dominant species in a temperate steppe ecosystem. Sci. Rep. 7, 40951–40961 (2017).
2. Kolodziejek, J. Effectof seed position and soil nutrients on seed mass, germination and seedling growth in Peucedanum oreoselinum
(Apiaceae). Sci. Rep. 7, 1959–1969 (2017).
3. van Mölken, T., Jorritsma-Wienk, L. D., van Hoek, P. H. W. & de Kroon, H. Only seed size matters for germination in different
populations of the dimorphic Tragopogon pratensis subsp. pratensis (Asteraceae). Am. J.Bot. 92, 432–437 (2005).
4. Violle, C., Castro, H., Richarte, J. & Navas, M. Intraspecific seed trait variations and competition: passive or adaptive response? Func.
Eco. 23, 612–620 (2009).
5. Zhi, X. et al. Effects of plant density on cotton yield components and quality. J. Integrat. Agri. 15, 1469–1479 (2016).
6. Sawan, Z. M., Fahmy, A. H. & Yousef, S. E. Direct and residual effects of nitrogen fertilization, foliar application of potassium and
plant growth retardant on Egyptian cotton growth, seed yield, seed viability and seedling vigor. Acta Eco. Sin. 29, 116–123 (2009).
7. Dordas, C. Foliar boron application affects lint and seed yield and improves seed quality of cotton grown on calcareous soils.
Nutrient Cycling Agroecosystems 76, 19–28 (2006).
8. Zhao, W. et al. Interactions of single mepiquat chloride application at different growth stages with climate, cultivar, and plant
population for cotton yield. Crop Sci. 57, 1–12 (2017).
9. Gwathmey, C. O. & Clement, J. D. Alteration of cotton source-sink relations with plant population density and mepiquat chloride.
Field Crops Res. 116, 101–107 (2010).
10. Wulff, R. D. Seed size variation in Desmodium paniculatum. I. Factors affecting seed size. J. Ecol. 74, 87–97 (1986).
11. Valencia-Díaz, S. & Montaña, C. Temporal variability in the maternal environment and its effect on seed size and seed quality in
Flourensia cernua DC. (Asteraceae). J. Arid Environ. 63, 686–695 (2005).
12. Balestri, E., Gobert, S., Lepoint, G. & Lardicci, C. Seed nutrient content and nutritional status of Posidonia oceanica seedlings in the
northwestern Mediterranean Sea. Marine Eco. Prog. Series 388, 99–109 (2009).
13. Dong, H., Kong, X., Li, W., Tang, W. & Zhang, D. Effects of plant density and nitrogen and potassium fertilization on cotton yield
and uptake of major nutrients in two fields with varying fertility. Field Crops Res. 119, 106–113 (2010).
14. Yang, F. et al. Plant growth regulation enhanced potassium uptake and use efficiency in cotton. Field Crops Res. 163, 109–118 (2014).
15. Yan, P. et al. A high plant density reduces the ability of maize to use soil nitrogen. PLOS one 12, e0172717 (2017).
16. Krannitz, P. G. Variation in magnesium and nitrogen content in seeds of Antelope bitterbrush (Purshia tridentata, Rosaceae). Am. J.
Bot. 84, 1738–1742 (1997).
17. Fenner, M. Environmental influences on seed size and composition. Horti. Rev. 74, 385–392 (1992).
18. Münzbergova, Z. Seed density significantly affects species richness and composition in experimental plant communities. PLoS One
7, e46704 (2012).
19. Yang, G., Luo, X., Nie, Y. & Zhang, X. Effects of plant density on yield and canopy micro environment in hybrid cotton. J. Integrat.
Agri. 13, 2154–2163 (2014).
20. Jiang, W. et al. Effects of narrow plant spacing on root distribution and physiological nitrogen use efficiency in summer maize. Crop
J. 1, 77–83 (2013).
21. Shorrocks, V. M. The occurrence and correction of boron deficiency. Plant Soil 193, 121–148 (1997).
22. Huang, L., Pant, J., Dell, B. & Bell, R. W. Effects of boron deficiency on anther development and floret fertility in wheat (Triticum
aestivum L. ‘Wilgoyne’). Ann. Bot. 85, 493–500 (2000).
23. Barker, A. V. & Pilbeam, D. J. Handbook of plant nutrition. (CRC press, Boca Raton, Florida, USA 2015).
24. Ahmed, N. et al. Impact of boron fertilization on dry matter production and mineral constitution of irrigated cotton. Pak. J. Bot. 43,
2903–2910 (2011).
25. Patel, M. S. & Golakiya, B. A. Effect of calcium carbonate and boron application on yield and nutrient uptake by groundnut. J. Ind.
Soc. Soil Sci. 34, 815–820 (1986).
26. Lopez-Lefebre, L. R. et al. Boron effect on mineral nutrients of tobacco. J. Plant Nutr. 25, 509–522 (2002).
27. Dursun, A. et al. Effects of boron fertilizer on tomato, pepper and cucumber yields and chemical composition. Commun. Soil Sci.
Plant Anal. 41, 1576–1593 (2010).
28. Sotiropoulos, T. E., Therios, N. I., Dimassi, N. K., Bosbalidis, A. & Kofilids, G. Nutritional status, growth, CO2 assimilation, and leaf
anatomical responses in two kiwi fruit species under boron toxicity. J. Plant Nutr. 5, 1244–1261 (2002).
29. Zhao, D. & Oosterhuis, D. M. Cotton growth and physiological responses to boron deficiency. J. Plant Nutr. 26, 856–867 (2003).
30. Dordas, C., Apostolides, G. E. & Goundra, O. Boron application affects seed yield and seed quality of sugar beets. J. Agric. Sci. 145,
377–384 (2007).
31. Khan, N. A. & Mobin, M. & Samiullah. The influence of gibberellic acid and sulfur fertilization rate on growth and S-use efficiency
of mustard (Brassica juncea). Plant Soil 270, 269–274 (2005).
32. Wang, R. et al. Exogenous application of brassinolide ameliorate chilling stress in Leymus chinensis (Trin.) Tzvel. by modulating
morphological, physiological and biochemical traits. Ban. J. Bot. 45, 143–150 (2016).
33. Song, J. et al. Combined foliar application of nutrients and 5-aminolevulinic acid (ALA) improved drought tolerance in Leymus
chinensis by modulating its morpho-physiological characteristics. Crop Past. Sci. 68, 474–482 (2017).
34. Nagel, O. W. & Lambers, H. Changes in the acquisition and partitioning of carbon and nitrogen in the gibberellin-deficient mutants
A70 and W335 of tomato (Solanum lycopersicum L.). Plant Cell Environ. 25, 883–891 (2002).
35. Agegnehu, G. & Taye, G. Effect of plant hormones on the growth and nutrient uptake of maize in acidic soils of the humid tropics.
SINET: Ethiop. J. Sci. 27, 17–24 (2004).
36. Wang, L. et al. The effect of mepiquat chloride on elongation of cotton (Gossypium hirsutum L.) internode is associated with low
concentration of gibberellic acid. Plant Sci. 225, 15–23 (2014).
37. Heilman, M. D. Effect of mepiquat chloride and nitrogen levels on yield, growth characteristics, and elemental composition of
cotton. J. Plant Growth Reg. 4, 41–47 (1985).
38. Zhang, S., Cothren, J. T. & Lorenz, E. J. Mepiquat chloride seed treatment and germination temperature effects on cotton growth,
nutrient partitioning, and water use efficiency. J. Plant Growth Reg. 9, 195–199 (1990).

ScIEntIfIc Reports | (2018) 8:7953 | DOI:10.1038/s41598-018-26308-5 13

www.nature.com/scientificreports/

39. Reddy, V. R., Trent, A. & Acock, B. Mepiquat chloride and irrigation versus cotton growth and development. Agron. J. 84, 930–933
(1992).
40. Zhao, D. & Oosterhuis, D. M. Pix plus and mepiquat chloride effects on physiology, growth, and yield of field-grown cotton. J. Plant
Growth Reg. 19, 415–422 (2000).
41. Gwathmey, C. O. & Craig, C. C. Managing earliness in cotton with mepiquat-type growth regulators. Crop Manag. 2, 1–8 (2003).
42. Nuti, R. C., Viator, R. P., Casteel, S. N., Edmisten, K. L. & Wells, R. Effect of planting date, mepiquat chloride, and glyphosate
application to glyphosate-resistant cotton. Agron. J. 98, 1627–1633 (2006).
43. Abbas, G. et al. Cotton response to multiple application of growth inhibitor (Mepiquat chloride). Pak. J. Agric. Sci. 47, 195–199
(2010).
44. Ren, X. et al. Managing mepiquat chloride and plant density for optimal yield and quality of cotton. Field Crops Res. 149, 1–10
(2013).
45. Mao, L. et al. Yield components and quality of intercropped cotton in response to mepiquat chloride and plant density. Field Crops
Res. 179, 63–71 (2015).
46. Jin, Z. Y., Yang, B. F. & He, Z. P. Study on the effects of DPC on cotton physiological function by isotope tracer. Acta Agri. Uni.
Pekinensis 10, 245–253 (in Chinese) (1984).
47. Duan, L. et al. Effects of mepiquat chloride on lateral roots initiation of cotton seedling and its mechanism. (In Proceedings:
Australian Agronomy Conference, Brisbane 2004).
48. Newman, E. I. & Andrews, R. E. Uptake of phosphorus and potassium in relation to root growth and root density. Plant Soil 38,
49–69 (1973).
49. Farooq, M., Atique-ur-Rehman, Aziz, T. & Habib, M. Boron nutripriming improves the germination and early seedling growth of
rice (Oryza sativa L.). J. Plant Nutr. 34, 1507–1515 (2011).
50. Wang, H. Y. & Chen, Y. A study with 32P on the effect of growth regulators on the distribution of nutrients with cotton plants. China
Cottons 4, 29–30 (1984).
51. Schon, M., Novacky, A. & Blevins, D. Boron induces hyperpolarization of sunflower root cell membranes and increases membrane
permeability to K+. Plant Physiol. 93, 566–571 (1990).
52. Ruiz, J. M., Baghour, M., Bretones, G., Belakir, A. & Romero, L. Nitrogen metabolism in tobacco plants (Nicotiana tabacum L.): role
of boron as a possible regulatory factor. Int. J. Plant Sci. 159, 121–126 (1998).
53. Mouhtaridou, G. N., Sotiropoulos, T. E., Dimassi, K. N. & Therios, I. N. Effects of boron on growth, and chlorophyll and mineral
contents of shoots of the apple rootstock MM 106 cultured in vitro. Biol. Plant. 48, 617–619 (2004).
54. Tungate, K. D., Susko, D. J. & Rufty, T. W. Reproduction and offspring competitiveness of Senna obtusifolia are influenced by nutrient
availability. New Phytol. 154, 661–669 (2002).
55. Yanlong, H. et al. Seed size effect on seedling growth under different light conditions in the clonal herb Ligularia virgaurea in
Qinghai-Tibet plateau. Acta Eco. Sin. 27, 3091–3108 (2007).
56. Ninh, T. P., Hiroshi, N. & Toru, T. Effectof umbel order and umbellet position on the production and characteristics of seeds and on
the development and growth of seedlings in Angelica acutiloba Kitagawa. Jap. J. Trop. Agri. 51, 46–53 (2007).
57. Parrish, J. A. D. & Bazzaz, F. A. Nutrient content of Abutilon theophrasti seeds and the competitive ability of the resulting plants.
Oecologia 65, 247–251 (1985).
58. Lacey, E., Smith, S. & Case, A. L. Parental effects on seed mass: seed coat but not seed embryo/endosperm effects. Am. J. Bot. 84,
1617–1620 (1997).
59. Estefan, G., Sommer, R. & Ryan, J. Methods of Soil, Plant, and Water Analysis: A Manual for the West Asia and North Africa Region.
(ICARDA, Beirut, Lebanon 2013).
60. Chapman, H. D. & Pratt, P. F. Methods of analysis for soils, plants and water. (University of California, Berkeley, CA, USA 1961).
61. Bingham, F. T. B. In: Methods of Soil Analysis, Part 2: Chemical and mineralogical properties. (ed Page, A. L.), 431–448 (American
Society of Agronomy, Madison, WI, USA. 1982).
62. Association of Official Seed Analysts. Rules for testing seeds. J. Seed Tech. 12, 1–112 (1990).
63. Ellis, R. A. & Roberts, E. H. The quantification of ageing and survival in orthodox seeds. Seed Sci. Tech. 9, 373–409 (1981).
64. Abdul-Baki, A. A. & Anderson, J. D. Vigour determination in soybean by multiple criteria. Crop Sci. 13, 630–637 (1973).

Acknowledgements
The authors are thankful to Higher Education Commission of Pakistan for financial support provided under
indigenous fellowship program to conduct this research.

Author Contributions
Conceived and designed experiments: A.Z. Performed experiments: A.Z., T.T. Analyzed the data: A.Z., T.T., M.N.,
T.R. Paper write up and improvement: A.Z., T.T., S.A.A., T.A., M.K., A.M., S.I., N.F., I.R.N., M.N. Supervised the
study: A.J., R.A.

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